HIV transmission via breast milk remains a significant mode of infant HIV transmission in the developing world. Interestingly, the majority of infants remain protected from breast milk transmission despite repeated low-dose exposures to the virus, with postnatal transmission occurring in only 10% of HIV-exposed, breastfed infants. This low rate of transmission raises the possibility that HIV-specific immunity in breast milk may protect infants from HIV transmission. We hypothesize that the evolution of compartmentalized virus quasispecies in breast milk is driven by virus-specific, local breast milk immunity. Using the rhesus monkey/SIV model of HIV pathogenesis, we will characterize cellular and humoral virus-specific immune responses and virus co-evolution in breast milk during acute and chronic SIV-infection. We will then investigate whether vaccination with a potent T cell-based vaccine regimen can elicit virus-specific immunity in breast milk. Uninfected, lactating Mamu-A*01+ rhesus monkeys will be vaccinated with plasmid DNA expressing the SIV genes gag, pol, and env, and boosted with a recombinant adenovirus vector expressing SIV gag, pol, and env. Vaccine-elicited SIV-specific cellular and humoral immune responses in breast milk will be examined throughout the vaccination schedule. Mucosal and systemic routes of vaccination will be compared for their ability to induce SIV-specific immune responses in breast milk. Finally, Mamu-A*01-H chronically SIV-infected, lactating rhesus monkeys will be vaccinated with the DNA prime/adenovirus boost regimen by the route that best elicited SIV-specific immune responses in the breast milk of uninfected rhesus monkeys. SIV-specific immune responses and virus replication kinetics in breast milk and blood will be examined in the chronically SIV-infected, lactating rhesus monkeys to determine if vaccine-elicited immune responses can control virus replication in breast milk. These studies will define the impact of virus-specific immunity in breast milk on local virus replication, and will provide a framework for the design of a maternal vaccine as a potential intervention to decrease breast milk transmission of HIV.